High throughput nucleic acid sequencing by expansion

• US 9,920,386 B2
• Filed: 07/06/2015
• Issued: 03/20/2018
• Est. Priority Date: 06/19/2007
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Stratos Genomics Inc.
• Inventors: Kokoris, Mark Stamatios, McRuer, Robert N.
• Primary Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US14/792,402
• Publication Number:
  US 20160208344A1
• Time in Patent Office: 988 Days
• Field of Search: None
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

Nucleic acid sequencing methods and related products are disclosed. Methods for sequencing a target nucleic acid comprise providing a daughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand, the Xpandomer comprising the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Corresponding products, including Xpandomers and oligomeric and monomeric substrate constructs are also disclosed.

Claims

1. A monomer substrate construct for use in a template-directed synthesis for sequencing a target nucleic acid having the following structure:

4. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises (i).

5. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises (ii).

6. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises both (i) and (ii).

7. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises an aqueous-soluble polymer.

8. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises a cationic polymer.

9. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises a polynucleotide.

10. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises a polyethylene glycol polymer.

11. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises an aliphatic polymer.

12. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises a cationic polymer, a polyethylene glycol polymer, an aliphatic polymer, a polynucleotide, or a combination thereof.

13. The monomer construct of claim 1, the duplex daughter strand of claim 2, or the Xpandomer of claim 3, wherein the tether further comprises one or more of each of the following:

a cationic polymer, a polyethylene glycol polymer, an aliphatic polymer, and a polynucleotide.

14. The monomer construct of claim 1 or the duplex daughter strand of claim 2, wherein the selectively cleavable bond is a phosphoramidate bond.

15. The monomer construct of claim 1 or the duplex daughter strand of claim 2, wherein the first end of the tether is attached to a heterocycle of the single nucleobase residue.

16. The monomer construct of claim 1 or the duplex daughter strand of claim 2, wherein the second end of the tether is attached to a mixed backbone of the single nucleobase residue.

17. The monomer construct of claim 1, wherein the end groups are independently hydroxyl, triphosphate or a mixed backbone.

2. A duplex daughter strand for sequencing a target nucleic acid having following structure:

20. The duplex daughter strand of claim 2, further comprising an end modifying group attached to the single nucleobase residue at the 3′

or 5′

end of the chain of m subunits, wherein the end modifying group comprises a charge or hydrophobicity property, or a combination thereof, capable of influencing the structure, position or rate, or combination thereof, at which an Xpandomer generated from the daughter strand is presented to the nanopore for detection.

30. The duplex daughter strand of claim 20, wherein the end modifying group is attached to the single nucleobase residue at the 3′

end of the chain of m subunits.

31. The duplex daughter strand of claim 20, wherein the end modifying group is attached to the single nucleobase residue at the 5′

end of the chain of m subunits.

3. An Xpandomer for sequencing a target nucleic acid having the following structure:

18. The Xpandomer of claim 3, wherein n¹ comprises a heterocycle portion of the single nucleobase residue.

19. The Xpandomer of claim 3, wherein n² comprises a backbone portion of the single nucleobase residue.

21. The Xpandomer of claim 3, further comprising an end modifying group attached to the n¹ or n² portion of the single nucleobase residue at the 3′

or 5′

end of the Xpandomer, wherein the end modifying group comprises a charge or hydrophobicity, or a combination thereof, capable of influencing the structure, position or rate, or combination thereof, at which the Xpandomer is presented to the nanopore for detection.

22. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group comprises a microbead, nanoparticle, nanocrystal, polymer, branched polymer, protein or dendrimer, or a combination thereof.

23. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group comprises a polymer.

24. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group produces a drag force that provides for elongation of the Xpandomer.

25. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group serves to pull the Xpandomer strand towards and through the nanopore.

26. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group comprises a charge property capable of influencing the position or rate, or combination thereof, at which the Xpandomer is presented to the nanopore for detection.

27. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group comprises a hydrophobicity property capable of influencing the structure, position or rate, or combinations thereof, at which the Xpandomer is presented to the nanopore for detection.

28. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group comprises:

(1) a charge property capable of influencing the position or rate, or combination thereof, at which the Xpandomer is taken up by the nanopore for detection, and (2) a hydrophobicity property capable of influencing the structure, position or rate, or combinations thereof, at which the Xpandomer is presented to the nanopore for detection.

29. The duplex daughter strand of claim 20 or the Xpandomer of claim 18, wherein the end modifying group comprises one or more nucleic acids.

32. The Xpandomer of claim 18, wherein the end modifying group is attached to the n¹ or n² portion of the single nucleobase residue at the 3′

end of the Xpandomer.

33. The Xpandomer of claim 18, wherein the end modifying group is attached to the n¹ or n² portion of the single nucleobase residue at the 5′

end of the Xpandomer.